Somatic mutations in clonally expanded cytotoxic T lymphocytes in patients with newly diagnosed rheumatoid arthritis.

Somatic mutations contribute to tumorigenesis. Although these mutations occur in all proliferating cells, their accumulation under non-malignant conditions, such as in autoimmune disorders, has not been investigated. Here, we show that patients with newly diagnosed rheumatoid arthritis have expanded CD8+ T-cell clones; in 20% (5/25) of patients CD8+ T cells, but not CD4+ T cells, harbour somatic mutations. In healthy controls (n=20), only one mutation is identified in the CD8+ T-cell pool. Mutations exist exclusively in the expanded CD8+ effector-memory subset, persist during follow-up, and are predicted to change protein functions. Some of the mutated genes (SLAMF6, IRF1) have previously been associated with autoimmunity. RNA sequencing of mutation-harbouring cells shows signatures corresponding to cell proliferation. Our data provide evidence of accumulation of somatic mutations in expanded CD8+ T cells, which may have pathogenic significance for RA and other autoimmune diseases.

Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML.

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3.

Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

Monitoring therapy responses at the leukemic subclone level by ultra-deep amplicon resequencing in acute myeloid leukemia.

In our individualized systems medicine program, personalized treatment options are identified and administered to chemorefractory acute myeloid leukemia (AML) patients based on exome sequencing and ex vivo drug sensitivity and resistance testing data. Here, we analyzed how clonal heterogeneity affects the responses of 13 AML patients to chemotherapy or targeted treatments using ultra-deep (average 68 000 × coverage) amplicon resequencing. Using amplicon resequencing, we identified 16 variants from 4 patients (frequency 0.54-2%) that were not detected previously by exome sequencing. A correlation-based method was developed to detect mutation-specific responses in serial samples across multiple time points. Significant subclone-specific responses were observed for both chemotherapy and targeted therapy. We detected subclonal responses in patients where clinical European LeukemiaNet (ELN) criteria showed no response. Subclonal responses also helped to identify putative mechanisms underlying drug sensitivities, such as sensitivity to azacitidine in DNMT3A mutated cell clones and resistance to cytarabine in a subclone with loss of NF1 gene. In summary, ultra-deep amplicon resequencing method enables sensitive quantification of subclonal variants and their responses to therapies. This approach provides new opportunities for designing combinatorial therapies blocking multiple subclones as well as for real-time assessment of such treatments.

Idelalisib sensitivity and mechanisms of disease progression in relapsed TCF3-PBX1 acute lymphoblastic leukemia.

TCF3-PBX1 (E2A-PBX1) is a recurrent gene fusion in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), which is caused by the translocation t(1;19)(q23;p13). TCF3-PBX1 BCP-ALL patients typically benefit from chemotherapy; however, many relapse and subsequently develop resistant disease with few effective treatment options. Mechanisms driving disease progression and therapy resistance have not been studied in TCF3-PBX1 BCP-ALL. Here, we aimed to identify novel treatment options for TCF3-PBX1 BCP-ALL by profiling leukemia cells from a relapsed patient, and determine molecular mechanisms underlying disease pathogenesis and progression. By drug-sensitivity testing of leukemic blasts from the index patient, control samples and TCF3-PBX1 positive and negative BCP-ALL cell lines, we identified the phosphatidylinositide 3-kinase delta (p110δ) inhibitor idelalisib as an effective treatment for TCF3-PBX1 BCP-ALL. This was further supported by evidence showing TCF3-PBX1 directly regulates expression of PIK3CD, the gene encoding p110δ. Other somatic mutations to TP53 and MTOR, as well as aberrant expression of CXCR4, may influence additional drug sensitivities specific to the index patient and accompanied progression of the disease. Our results suggest that idelalisib is a promising treatment option for patients with TCF3-PBX1 BCP-ALL, whereas other drugs could be useful depending on the genetic context of individual patients.

HOX gene expression predicts response to BCL-2 inhibition in acute myeloid leukemia.

Inhibitors of B-cell lymphoma-2 (BCL-2) such as venetoclax (ABT-199) and navitoclax (ABT-263) are clinically explored in several cancer types, including acute myeloid leukemia (AML), to selectively induce apoptosis in cancer cells. To identify robust biomarkers for BCL-2 inhibitor sensitivity, we evaluated the ex vivo sensitivity of fresh leukemic cells from 73 diagnosed and relapsed/refractory AML patients, and then comprehensively assessed whether the responses correlated to specific mutations or gene expression signatures. Compared with samples from healthy donor controls (nonsensitive) and chronic lymphocytic leukemia (CLL) patients (highly sensitive), AML samples exhibited variable responses to BCL-2 inhibition. Strongest CLL-like responses were observed in 15% of the AML patient samples, whereas 32% were resistant, and the remaining exhibited intermediate responses to venetoclax. BCL-2 inhibitor sensitivity was associated with genetic aberrations in chromatin modifiers, WT1 and IDH1/IDH2. A striking selective overexpression of specific HOXA and HOXB gene transcripts were detected in highly BCL-2 inhibitor sensitive samples. Ex vivo responses to venetoclax showed significant inverse correlation to ß2-microglobulin expression and to a lesser degree to BCL-XL and BAX expression. As new therapy options for AML are urgently needed, the specific HOX gene expression pattern can potentially be used as a biomarker to identify venetoclax-sensitive AML patients for clinical trials.

Safety and efficacy of the combination of pegylated interferon-α2b and dasatinib in newly diagnosed chronic-phase chronic myeloid leukemia patients.

Dasatinib (DAS) and interferon-α have antileukemic and immunostimulatory effects and induce deep responses in chronic myeloid leukemia (CML). We assigned 40 newly diagnosed chronic-phase CML patients to receive DAS 100 mg o.d. followed by addition of pegylated interferon-α2b (PegIFN) after 3 months (M3). The starting dose of PegIFN was 15 µg/week and it increased to 25 µg/week at M6 until M15. The combination was well tolerated with manageable toxicity. Of the patients, 84% remained on PegIFN at M12 and 91% (DAS) and 73% (PegIFN) of assigned dose was given. Only one patient had a pleural effusion during first year, and three more during the second year. After introduction of PegIFN we observed a steep increase in response rates. Major molecular response was achieved in 10%, 57%, 84% and 89% of patients at M3, M6, M12 and M18, respectively. At M12, MR(4) was achieved by 46% and MR(4.5) by 27% of patients. No patients progressed to advanced phase. In conclusion, the combination treatment appeared safe with very promising efficacy. A randomized comparison of DAS±PegIFN is warranted.

European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia.

Most reports on chronic myeloid leukaemia (CML) treatment with tyrosine kinase inhibitors (TKIs) focus on efficacy, particularly on molecular response and outcome. In contrast, adverse events (AEs) are often reported as infrequent, minor, tolerable and manageable, but they are increasingly important as therapy is potentially lifelong and multiple TKIs are available. For this reason, the European LeukemiaNet panel for CML management recommendations presents an exhaustive and critical summary of AEs emerging during CML treatment, to assist their understanding, management and prevention. There are five major conclusions. First, the main purpose of CML treatment is the antileukemic effect. Suboptimal management of AEs must not compromise this first objective. Second, most patients will have AEs, usually early, mostly mild to moderate, and which will resolve spontaneously or are easily controlled by simple means. Third, reduction or interruption of treatment must only be done if optimal management of the AE cannot be accomplished in other ways, and frequent monitoring is needed to detect resolution of the AE as early as possible. Fourth, attention must be given to comorbidities and drug interactions, and to new events unrelated to TKIs that are inevitable during such a prolonged treatment. Fifth, some TKI-related AEs have emerged which were not predicted or detected in earlier studies, maybe because of suboptimal attention to or absence from the preclinical data. Overall, imatinib has demonstrated a good long-term safety profile, though recent findings suggest underestimation of symptom severity by physicians. Second and third generation TKIs have shown higher response rates, but have been associated with unexpected problems, some of which could be irreversible. We hope these recommendations will help to minimise adverse events, and we believe that an optimal management of them will be rewarded by better TKI compliance and thus better CML outcomes, together with better quality of life.

Frontline nilotinib in patients with chronic myeloid leukemia in chronic phase: results from the European ENEST1st study.

The Evaluating Nilotinib Efficacy and Safety in Clinical Trials as First-Line Treatment (ENEST1st) study included 1089 patients with newly diagnosed chronic myeloid leukemia in chronic phase. The rate of deep molecular response (MR(4) (BCR-ABL1⩽0.01% on the International Scale or undetectable BCR-ABL1 with ⩾10,000 ABL1 transcripts)) at 18 months was evaluated as the primary end point, with molecular responses monitored by the European Treatment and Outcome Study network of standardized laboratories. This analysis was conducted after all patients had completed 24 months of study treatment (80.9% of patients) or discontinued early. In patients with typical BCR-ABL1 transcripts and ⩽3 months of prior imatinib therapy, 38.4% (404/1052) achieved MR(4) at 18 months. Six patients (0.6%) developed accelerated or blastic phase, and 13 (1.2%) died. The safety profile of nilotinib was consistent with that of previous studies, although the frequencies of some nilotinib-associated adverse events were lower (for example, rash, 21.4%). Ischemic cardiovascular events occurred in 6.0% of patients. Routine monitoring of lipid and glucose levels was not mandated in the protocol. These results support the use of frontline nilotinib, particularly when achievement of a deep molecular response (a prerequisite for attempting treatment-free remission in clinical trials) is a treatment goal.

Novel drug candidates for blast phase chronic myeloid leukemia from high-throughput drug sensitivity and resistance testing.

Chronic myeloid leukemia in blast crisis (CML BC) remains a challenging disease to treat despite the introduction and advances in tyrosine kinase inhibitor (TKI) therapy. In this study we set out to identify novel candidate drugs for CML BC by using an unbiased high-throughput drug testing platform. We used three CML cell lines representing different types of CML blast phases (K562, EM-2 and MOLM-1) and primary leukemic cells from three CML BC patients. Profiling of drug responses was performed with a drug sensitivity and resistance testing platform comprising 295 anticancer agents. Overall, drug sensitivity scores and the drug response profiles of cell line and primary cell samples correlated well and were distinct from other types of leukemia samples. The cell lines were highly sensitive to TKIs and the clinically TKI-resistant patient samples were also resistant ex vivo. Comparison of cell line and patient sample data identified new candidate drugs for CML BC, such as vascular endothelial growth factor receptor and nicotinamide phosphoribosyltransferase inhibitors. Our results indicate that these drugs in particular warrant further evaluation by analyzing a larger set of primary patient samples. The results also pave way for designing rational combination therapies.

Autologous stem cell transplantation versus novel drugs or conventional chemotherapy for patients with relapsed multiple myeloma after previous ASCT.

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common first-line treatment for patients with multiple myeloma (MM) under 65 years of age. A second ASCT at first relapse is frequently used but is challenged by the use of novel drugs. We retrospectively studied the outcome of second-line treatment in MM patients from the Nordic countries with relapse after first-line HDT and ASCT. Patients that underwent a second ASCT (n=111) were compared with patients re-treated with conventional cytotoxic drugs only (n=91) or with regimens including novel drugs (proteasome inhibitors and/or immunomodulatory drugs) (n=362) without a second ASCT. For patients receiving a second ASCT median overall survival was 4.0 years compared with 3.3 years (P<0.001) for the group treated with novel drugs and 2.5 years (P<0.001) for those receiving conventional cytotoxic drugs only. A second ASCT also resulted in a significantly longer second time to progression and a significantly longer time to next treatment. We conclude that, irrespective of the addition of novel drugs, MM patients in first relapse after ASCT still appear to benefit from a second ASCT. A second ASCT should be considered for all physically fit patients.

Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation.

T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene.

Impact of malignant stem cell burden on therapy outcome in newly diagnosed chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38-) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1-100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P=0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P=0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

Rapid mobilization of cytotoxic lymphocytes induced by dasatinib therapy.

Tyrosine kinase inhibitors (TKIs) have potent effects on malignant cells, and they also target kinases in normal cells, which may have therapeutic implications. Using a collection of 55 leukemia patients treated with TKI therapy (chronic myeloid leukemia, n=47; acute lymphoblastic leukemia, n=8), we found that dasatinib, a second-generation broad-spectrum TKI, induced a rapid, dose-dependent and substantial mobilization of non-leukemic lymphocytes and monocytes in blood peaking 1-2 h after an oral intake and the blood counts closely mirrored drug plasma concentration. A preferential mobilization was observed for natural killer (NK), NK T, B and γδ+ T cells. Mobilization was coupled with a more effective transmigration of leukocytes through an endothelial cell layer and improved cytotoxicity of NK cells. Platelet numbers decreased markedly after the drug intake in a proportion of patients. Similar effects on blood cell dynamics and function were not observed with any other TKI (imatinib, nilotinib and bosutinib). Thus, dasatinib induces a unique, rapid mobilization and activation of cytotoxic, extravasation-competent lymphocytes, which may not only enhance antileukemia immune responses but can also be causally related to the side-effect profile of the drug (pleural effusions, thrombocytopenia).

Expansion of highly differentiated CD8+ T-cells or NK-cells in patients treated with dasatinib is associated with cytomegalovirus reactivation.

The tyrosine kinase inhibitor dasatinib exerts immunosuppressive effects on T-cells and NK-cells in vitro. However, in some dasatinib-treated leukemia patients, clonal lymphocytosis with large granular lymphocyte (LGL) morphology develops, and this is associated with enhanced therapeutic responses. To elucidate the mechanistic basis for this paradoxical observation, we conducted detailed phenotypic and functional analyses of T-cell and NK-cell populations from 25 dasatinib-treated leukemia patients. All tested patients with LGL expansions (15/16) were cytomegalovirus (CMV) immunoglobulin (IgG) seropositive with high frequencies of CMV-specific CD8(+) T-cells; 5/16 LGL patients also experienced symptomatic CMV reactivation during dasatinib therapy. Expanded T-cell and NK-cell populations exhibited late differentiated (CD27(-)CD57(+)) phenotypes; this was associated with a predisposition to apoptosis within the T-cell compartment and impaired NK-cell cytotoxicity. Only 3/9 non-LGL patients were CMV IgG seropositive. Dasatinib inhibited in vitro lymphocyte functions, similarly in LGL patients and controls. Notably, distinct CD8(high) and CD8(low) T-cell subsets were observed in LGL patients; this phenotypic dichotomy was also apparent in CMV-specific CD8(+) T-cell populations, and exhibited features consistent with antigen-driven activation. In addition, plasma levels of IP-10, IL-6, monokine induced by interferon-γ and interleukin-2R were significantly increased in LGL patients. These data provide evidence that dasatinib-associated LGL expansion is linked to CMV reactivation and suggest a potential mechanism for this phenomenon.